(LCAT) is an enzyme that is responsible for the synthesis of cholesteryl esters (CEs) in plasma. During the last grant cycle, we demonstrated that LCAT expression is inversely associated with atherosclerosis development in low density lipoprotein receptor knockout (LDLr -/-) mice and that this response was independent of plasma lipid concentrations. The overall goal of this grant renewal is to understand the molecular mechanisms through which LCAT protects against atherosclerosis development. We will explore a novel hypothesis that LCAT trafficking of oxidized fatty acids (FAs) in lipoprotein phosphatidylcholine (PC) will reduce atherosclerosis by removal of oxidized short chained FA groups via PLA2 cleavage and by lipoprotein particle core enrichment of minimally oxidized FAs (hydroxyoctadecadienoic acid, HODE and hydroperoxyoctadecadienoic acid, HPODE) that are ligands for PPAR-gamma, by transesterification to cholesterol. In specific aim 1, recombinant lipoprotein particles containing apoA-I, cholesterol, and PC with varying amounts of well-defined oxidized long and short chain PC species will be made, chemically analyzed and reacted with purified human recombinant LCAT. LCAT kinetics will be measured and reaction products will be identified and quantified using mass spectrometry (MS) analysis. In specific aim 2, we will test the hypothesis that LCAT protects against atherosclerosis by decreasing the atherogenicity of plasma LDL. Mice expressing different levels of LCAT and fed atherogenic diets containing saturated or n-6 polyunsaturated fat will be used to harvest LDL to identify and quantify oxidized PC and CE by mass spectrometry (MS) and to assess the ability of LDLs to activate cultured endothelial cells and macrophages. Aortas will be harvested to quantify oxidized PC and CE by MS and to determine expression of genes involved in atherogenesis. In specific aim 3, we will extend our preliminary finding that LCAT is a major contributor of CE in plasma LDL particles, and test two hypotheses: 1) that LCAT activity on LDL particles is activated by apolipoprotein E, and 2) that LDL particles generated by LCAT will be less atherogenic compared to those generated by acyl CoA:cholesterol acyltransferase-2. If our hypothesis is correct, it could help explain the well-documented paradox that oxidized lipids contribute to the pathogenesis of atherosclerosis, but diets enriched in polyunsaturated fatty acids, which are more easily oxidized than saturated and monounsaturated fatty acids, result in less, not more atherosclerosis.